Non-Reducing End Unsaturated Mannuronic Acid Oligosaccharides And Compositions Containing Same As Active Ingredient

ABSTRACT

Disclosed is a non-reducing end unsaturated mannuronic acid oligosaccharide having a molecular weight of 100-3000 Da, which is obtained by lysing polymannuronate as a substrate with alginate lyase, and provided are: a non-reducing end unsaturated mannuronic acid oligosaccharide; and pharmaceutical compositions for alleviating, preventing, or treating obesity, diabetes, and climacteric syndrome, and probiotics for promoting intestinal beneficial bacteria, the compositions and probiotics containing, as an active ingredient, the non-reducing end unsaturated mannuronic acid oligosaccharide, so that the antiobesity and antidiabetic effects, estrogen activity, and intestinal microflora controlling effect of the non-reducing end unsaturated mannuronic acid oligosaccharides are remarkably excellent as compared with non-reducing end saturated mannuronic acid oligosaccharides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of Korean PatentApplication No. 10-2015-0043404, filed 27 Mar. 2015. The entiredisclosure of the above applications is incorporated herein byreferences.

FIELD

The present disclosure relates to non-reducing end unsaturatedmannuronic acid oligosaccharides and compositions containing the same asan active ingredient.

BACKGROUND

In the modern world, the functional food market is expanding with theincrease in complex metabolic syndromes caused by obesity (based on theyear 2014, 18% of OECD adults) and diabetes (based on the year 2011,6.9%).

The metabolic syndrome refers to the complex occurrence of obesity, type2 diabetes caused by insulin resistance, and various metabolicabnormalities. The metabolic syndromes rapidly increase due to the agingpopulation and high-calorie diets habits, causing social costs, and thusthe prevention of fundamental causes and the development of medical orfood materials are urgent.

In the human intestinal ecosystem, microbes are present at birth tomaintain the balance between beneficial bacteria and harmful bacteria.The microbes form intestinal microflora, coexist with humans, and have adirect or indirect effect on human health through interactions withhumans. Recently, the National Institutes of Health (NIH) researched therelationships between intestinal microflora and diseases through the“Human Microbiome Project”, and raised the importance of thenormalization of the intestinal flora since having unbalanced microbialflora causes the occurrence of inflammatory enteric diseases and thelike.^(1,2)

According to the paper about the association between obesity and gutmicrobiome, which was published on the scientific journal “Nature” in2006, slim people are different from obese people with respect to gutmicrobiome distribution, and it was confirmed through an experimentusing mice that bacteria belonging to the Firmicutes show a relativelyhigher component percentage than bacteria belonging to the Bacteroidetesin obese mice.³ Since then, the research about intestinal microbes andthe human body has been conducted in various fields, and the developmentof intestinal microflora improving preparations is needed for thesuppression of obesity and the treatment of diseases through theimprovement in intestinal microflora.

Approximately 500,000 species of marine organisms, which correspond toabout 80% of all species on Earth, are assumed to exist. However, ofthese, less than 1% of the marine organisms are being developed asuseful living resources, and thus have a very high developmentpotential. Alginic acid, which is a representative seaweedpolysaccharide as a seaweed-derived functional material, is contained in15-35% of brown algae, such as kelp or seaweed, and has polyuronidecharacteristics, in which two kinds of uronic acids, β-D-mannuronic acid(M) and α-L-guluronic acid (G), are connected via 1,4-glycosidiclinkages at various ratios. Alginic acid-derived oligosaccharides may beclassified into mannuro-oligosaccharide (MOS), guluro-oligosaccharide(GOS), and mannuronate and guluronate mixed oligosaccharide (alginateoligosaccharide, AOS), according to the component sugar, and may also beclassified according to the double bond at the end of sugar.

Marine-derived polysaccharides have been used in human life for a longtime, and the research about biological activities, such as anticanceractivity, antioxidation, antihypertension, and antibiotic materials,which are derived from marine organisms, are being conducted activelyand globally. The output of alginic acid sources produced globally isapproximately 100,000 tons, of which about 30% are used as a foodadditive, but when the alginic acid sources are developed as highvalue-added medicinal sources, the values thereof can be doubled. Theresearch of alginic acid-derived oligosaccharides achieves tangibleresults, such as being reported to have the biological activities ofpromoting the growth of roots of higher plants, promoting the growth ofBifidobacterium sp., anti-inflammation, antioxidation, and antibioticactivity, according to the structural feature, and thus alginicacid-derived oligosaccharides have widespread application fields.

Throughout the entire specification, many papers and patent documentsare referenced and their citations are represented. The disclosure ofthe cited papers and patent documents are entirely incorporated byreference into the present specification and the level of the technicalfield within which the present invention falls, and the details of thepresent invention are explained more clearly.

SUMMARY

The present inventors endeavored to promote the use of alginic acidoligosaccharides for food and medicine. As a result, the presentinventors investigated non-reducing end unsaturated mannuronic acidoligosaccharides, which are derived from alginic acid and have variousbiological activities, such as antiobesity and antidiabetic actions, animprovement in intestinal microflora, and estrogen efficacy, and thencompleted the present invention.

Therefore, an aspect of the present disclosure is to providenon-reducing end unsaturated mannuronic acid oligosaccharides.

Another aspect of the present disclosure is to provide compositions foralleviating, preventing, or treating obesity.

Still another aspect of the present disclosure is to providecompositions for alleviating, preventing, or treating diabetes.

Another aspect of the present disclosure is to provide probiotics forpromoting intestinal beneficial bacteria.

Still another aspect of the present disclosure is to providecompositions for alleviating, preventing, or treating climactericsyndrome.

Other purposes and advantages of the present disclosure will become moreobvious with the following detailed description of the invention,claims, and drawings.

In accordance with an aspect of the present invention, there is provideda non-reducing end unsaturated mannuronic acid oligosaccharide having amolecular weight of 100-3000 Da, which is obtained by lysingpolymannuronate as a substrate with alginate lyase.

The present inventors endeavored to promote the use of alginic acidoligosaccharides for food and medicine. As a result, the presentinventors investigated non-reducing end unsaturated mannuronic acidoligosaccharides, which are derived from alginic acid and have variousbiological activities, such as antiobesity and antidiabetic actions, animprovement in intestinal microflora, and estrogen efficacy.

As used herein, the term “non-reducing” refers to a feature of nothaving carbon of an anomer (a type of diastereomer in which a hydrogenatom and a hydroxyl group attached on one carbon atom are interchangedwith each other in a cyclic reaction generating hemiacetals (forming aring between C-1 and C-5) and hemiketals (forming a ring between C-2 andC-5) of monosaccharides)) in the oligosaccharide structure.

As used herein, the term “unsaturated” refers to a form in which acarbon chain with hydrogen atoms is unsaturated, and the term“saturated” refers to a form in which a carbon chain with hydrogen atomsis saturated.

The non-reducing end unsaturated mannuronic acid oligosaccharides of thepresent invention include all types of mannuronic acid oligosaccharides,which have no anomeric carbon and of which a carbon chain with hydrogenatoms is unsaturated.

The non-reducing end unsaturated mannuronic acid oligosaccharide hasstructural formula 1 shown in an example below (a mannuronic acidoligosaccharide having three linked sugars):

The non-reducing end unsaturated mannuronic acid oligosaccharide has aZ-average molecular weight (m/z) of 175 for one sugar, 351 for twosugars, 527.4 for four sugars, 880 for five sugars, and 1056 for sixsugars, and 1232 for seven sugars (FIG. 2)

As used herein, the term “non-reducing end saturated mannuronic acidoligosaccharide” refers to a saturated mannuronic acid oligosaccharideobtained by acid hydrolysis of polymannuronate.

The non-reducing end unsaturated mannuronic acid oligosaccharide isprepared by lysing polymannuronate as a substrate with alginate lyase.

According to an embodiment of the present invention, the alginate lyaserefers to an enzyme that lyses alginate, which is composed ofpolyguluronate and polymannuronate, into low molecules.

According to a specific embodiment of the present invention, thealginate lyase is AlyDW11 (Korean Patent Registration No. 10-1277706)derived from an abalone intestinal strain.

The non-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention is composed of one or more sugars.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide includes one to tenmannuronic acids or guluronic acids. According to another embodiment ofthe present invention, the non-reducing end unsaturated mannuronic acidoligosaccharide includes one to nine mannuronic acids or guluronicacids. According to still another embodiment of the present invention,the non-reducing end unsaturated mannuronic acid oligosaccharideincludes one to eight mannuronic acids or guluronic acids. According toa particular embodiment of the present invention, the non-reducing endunsaturated mannuronic acid oligosaccharide includes one to sevenmannuronic acids or guluronic acids.

The non-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention has a deletion of a water molecule, and thus has asmaller mass value by approximately 18, which corresponds to a massvalue of the water molecule, compared with the non-reducing endsaturated mannuronic acid oligosaccharide (FIG. 2).

The non-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention is composed of mannuronic acids and guluronic acids.

According to an embodiment of the present invention, the ratio ofmannuronic acids : guluronic acids is 1.2-5.0:1 in the non-reducing endunsaturated mannuronic acid oligosaccharide. According to anotherembodiment of the present invention, the ratio is 1.2-4.5:1. Accordingto still another embodiment of the present invention, the ratio is1.8-4.0:1. According to a particular embodiment of the presentinvention, the ratio is 2.0-3.0:1.

That is, the non-reducing end unsaturated mannuronic acidoligosaccharide more predominantly contains mannuronic acids rather thanguluronic acids by 1.2-5.0, 1.2-4.5, 1.8-4.0, or 2.0-3.0 times (FIG. 4).

In accordance with another aspect of the present invention, there isprovided a composition for alleviating, preventing, or treating obesity,the composition containing, as an active ingredient, the non-reducingend unsaturated mannuronic acid oligosaccharide.

As used herein, the term “obesity” refers to a condition in whichadipose tissues are excessively accumulated in the body so as to causehealth disorders.

The non-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention suppresses lipid accumulation.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide suppresses theaccumulation of triglycerides by 20-60%, 25-50%, or 30-40%, and thenon-reducing end saturated mannuronic acid oligosaccharide suppressesthe accumulation of triglycerides by 10-30%, 10-25%, or 10-30%.

According to another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide suppressesthe accumulation of triglycerides by at least two times, compared withthe non-reducing end saturated mannuronic acid oligosaccharide.

The non-reducing end unsaturated mannuronic acid oligosaccharidecontrols intestinal microflora inducing obesity.

As used herein, the term “intestinal microflora or gut microflora”refers to microorganism complex community growing in animal guts. Eachmicroorganism constituting intestinal microflora is beneficial orharmful to hosts due to material production or lytic ability, and forexample, the intestinal microflora, as a whole, is involved in providingvitamins, preventing infection and helping gut functions (peristalticmovement and absorption), and therefore, the composition of themicroflora is closely related to constipation and otherintestine-related diseases (Mistuoka T. Bifidobacteria Microflora, 1(1):3, 1982).

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide reduces the growth ofintestinal bacterial strains to control the intestinal microflora.

According to another embodiment of the present invention, the intestinalbacterial strains are selected from the group consisting of Roseburiasp. and Lactobacillus sp.

The non-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention inhibits the expression of adipocytedifferentiation-related genes inducing obesity.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide inhibits the expressionof adipocyte protein 2 (aP2), CAAT enhancer binding protein α (C/EBPα),and peroxisome proliferator-activated receptor γ (PPARγ).

According to another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide inhibitsthe expression of aP2, C/EBPα, and PPARγ by 15-35%, 50-70%, and 30-50%,respectively. This effect is superior to that of the non-reducing endsaturated mannuronic acid oligosaccharide by 1.5-7.0 times.

The composition for alleviating, preventing, or treating obesity of thepresent invention may be prepared as a pharmaceutical composition forpreventing or treating obesity, or a food composition or functional foodcomposition for alleviating or preventing obesity.

In accordance with still another aspect of the present invention, thereis provided a composition for alleviating, preventing, or treatingobesity, the composition containing, as an active ingredient, thenon-reducing end unsaturated mannuronic acid oligosaccharide.

As used herein, the term “diabetes” refers to a chronic diseasecharacterized by a relative or absolute shortage in insulin, causingglucose-intolerance. The term “diabetes” includes all types of diabetes,for example, type 1 diabetes, type 2 diabetes, or hereditary diabetes.Type 1 diabetes is the insulin-dependent diabetes, and is mainly causedby β-cell disruption. Type 2 diabetes is the insulin-independentdiabetes, and is caused by an insufficient secretion of insulin aftereating or by insulin resistance.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide promotes glucose uptake.

These effects of the present invention result from only the non-reducingend unsaturated mannuronic acid oligosaccharide, but are not exhibitedby the non-reducing end saturated mannuronic acid oligosaccharide.

According to another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide promotesglucose uptake via the AMP-activated protein kinase (AMPK) pathway.

The composition for alleviating, preventing, or treating diabetes of thepresent invention may be prepared as a pharmaceutical composition forpreventing or treating diabetes, or a food composition or functionalfood composition for alleviating or preventing diabetes.

In accordance with still another aspect of the present invention, thereis provided a probiotic for promoting intestinal beneficial bacteria,containing, as an active ingredient, the non-reducing end unsaturatedmannuronic acid oligosaccharide.

The probiotic for promoting intestinal beneficial bacteria of thepresent invention promotes the growth of intestinal beneficial bacteria.

In an embodiment of the present invention, the intestinal beneficialbacteria are selected from the group consisting of Roseburia sp. andLactobacillus sp.

As used herein, the term “probiotic” refers to a food supplementcontaining living bacteria, which helps for the health of a host organicbody. The probiotic composition of the present invention may be preparedas a fermented milk product, but may be prepared in the form of agranule, a powder, or the like.

The administration method of the probiotic composition of the presentinvention is not particularly limited, but the probiotic composition maybe orally administered in the form of a pill or a tablet, or may beadministered by being added to food in the form of a powder or agranule. For example, when used as a medicine, the composition of thepresent invention per se may be used without formulating each componentpowder, but may be formulated in the dosage form of a powder, a granule,a fine granule, a tablet, a sugar-coated tablet, a capsule, a tablet, anenteric-coated preparation, or the like. An excipient, a binder, adisintegrant, or the like, which is used in general medicinalpreparations, may be used as a diluent, and besides, a colorant, astabilizer, a preserver, a lubricant, or the like may be added. Whenused as food, the food composition of the present invention per se maybe used without formulating each component powder, but may be processedin a form that is suitable to uptake by adding a plant fiber, anoligosaccharide, a grain, a vitamin, and the like, or adding aflavoring, a colorant, a sweetening agent, and the like. In addition,the food composition, as a food additive, may be added and mixed withanother food.

The probiotic for promoting intestinal beneficial bacteria of thepresent invention may be prepared into a pharmaceutical composition, afood composition, or a functional food composition.

In accordance with another aspect of the present invention, there isprovided a composition for alleviating, preventing, or treatingclimacteric syndrome, the composition containing, as an activeingredient, the non-reducing end unsaturated mannuronic acidoligosaccharide.

As used herein, the term “climacteric syndrome” is a kind of femaleinternal secretion syndrome, and refers to a transition period of thereduction or loss of physiological and sexual functions through generaland gradual reductions of ovarian functions regardless of natural loss,loss of surgery, or chemically induced loss, and reaching the menopause,as one procedure during the climacteric period, which is a permanentstop of menstruation occurring after the ovarian functions are stopped.In the menopausal period, acute or chronic symptoms may occur dependingon hormone changes, such as the reduction in estrogen production, theincreases in follicle stimulating hormone and luteinizing hormone, etc.That is, vasomotor symptoms, such as hot flushes and night sweating, andpsychological symptoms, such as anxiety, lack of concentration, anddepression, may be shown as initial symptoms, and there may be urinaryreproductive system and skin symptoms within a few years of menopause,and there may be osteoporosis, cardiovascular, and cerebrovasculardiseases in few years after menopause.

The climacteric syndromes include a symptom selected from the groupconsisting of facial flushing, sweating, heart discomfort, sleepproblems, depression, irritability, anxiety, physical fatigue, mentalfatigue, sexual problems, urinary problems, vaginal dryness, jointdiscomfort, and muscle discomfort.

The composition containing the non-reducing end unsaturated mannuronicacid oligosaccharide as an active ingredient of the present inventionincreases the activity of estrogen.

The non-reducing end unsaturated mannuronic acid oligosaccharideactivates estrogen through the expression of estrogen response element(ERE)-mediated underlying genes via an estrogen receptor pathway, inwhich estrogen acts as a ligand, and an estrogen-related receptor apathway, which acts independently from estrogen.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide induces the expressionof ERE via estrogen receptor α.

According to another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide induces theexpression of ERE by expressing estrogen-related receptor α.

The activity of estrogen is increased through the expression ofunderlying estrogen genes due to the expression of ERE.

According to still another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide increasesthe mRNA expression of presenilin 2 (pS2), progesterone receptor (PR),and E2-mediated cathepsin D (CTSD).

According to another embodiment of the present invention, thenon-reducing end unsaturated mannuronic acid oligosaccharide increasesthe mRNA expression of peroxisome proliferator-activated receptor gammacoactivator 1-α (PGC-1α), estrogen-related receptor α (ERRα),trans-acting T-cell-specific transcription factor (GATA3), and forkheadbox protein A1 (FOXA1).

GATA3 and FOXA1 are important factors in mammary cell differentiation,and serve to inhibit the differentiation into cancer cells or malignanttumors.

The composition of the present invention further contains 17β-estradiol.

According to an embodiment of the present invention, the non-reducingend unsaturated mannuronic acid oligosaccharide shows a synergic effecttogether with 17β-estradiol.

The composition of the present invention may be prepared as apharmaceutical composition for preventing or treating climactericsyndrome, or a food composition or functional food composition foralleviating or preventing climacteric syndrome.

Here, (a) the composition for alleviating, preventing, or treatingobesity; (b) the composition for alleviating, preventing, or treatingdiabetes; (c) the probiotic for promoting intestinal beneficialbacteria; and (d) the composition for alleviating, preventing, ortreating climacteric syndrome, of the present invention, may be preparedinto a pharmaceutical composition.

According to a preferable embodiment of the present invention, thecomposition of the present invention contains: (a) a pharmaceuticallyeffective amount of the above-described non-reducing end unsaturatedmannuronic acid oligosaccharide of the present invention; and (b) apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically effective amount” refers to an amount that issufficient to attain the efficacy or activity of the above-describednon-reducing end unsaturated mannuronic acid oligosaccharide.

In cases where the composition of the present invention is prepared as apharmaceutical composition, the pharmaceutical composition of thepresent invention contains a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier contained in the pharmaceuticalcomposition of the present invention is conventionally used at the timeof formulation, and examples thereof may include, but are not limitedto, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum,calcium phosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and mineral oil. The pharmaceutical composition ofthe present invention may further contain a lubricant, a wetting agent,a sweetening agent, a flavoring agent, an emulsifier, a suspendingagent, a preservative, and the like, in addition to the aboveingredients. Suitable pharmaceutically acceptable carriers andpreparations are described in detail in Remington's PharmaceuticalSciences (19th ed., 1995).

The pharmaceutical composition of the present invention may beadministered orally or parenterally, and preferably, the oraladministration manner is employed.

A suitable dose of the pharmaceutical composition of the presentinvention may vary depending on various factors, such as the method forformulation, manner of administration, the age, body weight, gender, andmorbidity of the patient, diet, time of administration, excretion rate,and response sensitivity. A general dose of the pharmaceuticalcomposition of the present invention is within the range of 0.001μg/kg-100 mg/kg in adults.

The pharmaceutical composition of the present invention may beformulated into a unit or multiple dosages form using a pharmaceuticallyacceptable carrier and/or excipient according to the method easilyconducted by a person having an ordinary skill in the art to which thepresent invention pertains. Here, the dosage form may be a solution inan oily or aqueous medium, a suspension, a syrup, or an emulsion, anextract, a powder, a granule, a tablet, or a capsule, and may furtherinclude a dispersant or a stabilizer.

As used herein, the term “containing, as an active ingredient” refers tothe inclusion of an amount that is sufficient to attain the efficacy oractivity of the above-described non-reducing end unsaturated mannuronicacid oligosaccharide. The quantitative upper limit of theabove-described non-reducing end unsaturated mannuronic acidoligosaccharide contained in the composition of the present inventionmay be selected within an appropriate range by a person skilled in theart.

Here, (a) the composition for alleviating, preventing, or treatingobesity; (b) the composition for alleviating, preventing, or treatingdiabetes; (c) the probiotic for promoting beneficial bacteria; and (d)the composition for alleviating, preventing, or treating climactericsyndromes, of the present invention, may be prepared into a foodcomposition.

In cases where the composition containing, as an active ingredient, thenon-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention, is prepared into a food composition, it containscomponents that are generally added at the time of food making, besidesthe non-reducing end unsaturated mannuronic acid oligosaccharide, andcontains, for example, proteins, hydrocarbons, fats, nutrients,seasonings, and flavoring agents. Examples of the carbohydrate aremonosaccharides, such as glucose and fructose; disaccharides, such asmaltose, sucrose, and oligosaccharides; polysaccharides such as dextrin;typical sugars such as cyclodextrin; sugar alcohols, such as, xylitol,sorbitol, and erythritol. Examples of the flavoring agent may be naturalflavoring agents (thaumatin, and stevia extract (e.g., rebaudioside A,glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin,aspartame, etc.) For example, a drink, which is made from the foodcomposition, may further contain citric acid, liquefied fructose, sugar,glucose, acetic acid, malic acid, fruit juice, an extract of Eucommiaulmoides, a jujube extract, and an licorice extract, in addition to thenon-reducing end unsaturated mannuronic acid oligosaccharide of thepresent invention.

The composition of the present invention may be prepared as a functionalfood composition containing, as an active ingredient, the non-reducingend unsaturated mannuronic acid oligosaccharide of the presentinvention. The composition of the present invention, when prepared as afunctional food composition, contains components that are normally addedat the time of food making, for example, proteins, carbohydrates, fats,nutrients, seasoning, and flavoring agents. For example, the compositionof the present invention, when used as a drink, may contain a flavoringagent or a natural hydrocarbon as an additive component, in addition tothe non-reducing end unsaturated mannuronic acid oligosaccharide.Examples of the natural hydrocarbon include monosaccharides (e.g.,glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, etc.);oligosaccharides; polysaccharides (e.g., dextrin, cyclodextrin, etc.);and sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). As theflavoring agent, natural flavoring agents (e.g., thaumatin, steviaextract etc.) and synthetic flavoring agents (e.g., saccharin,aspartame, etc.,) may be used.

The non-reducing end unsaturated mannuronic acid oligosaccharide is anactive ingredient for biological activity of alginate, which is known inthe prior art, and exhibits an antiobesity effect, an antidiabeticeffect, an effect of improving climacteric syndrome, and an effect ofcontrolling intestinal microflora. These effects are remarkably superiorcompared with the non-reducing end saturated mannuronic acidoligosaccharide, and this means that the double bond at the end sugar ofthe non-reducing end saturated mannuronic acid oligosaccharide and theunsaturated form thereof are important factors.

Features and advantages of the present invention are summarized asfollows:

(a) The present invention provides non-reducing end unsaturatedmannuronic acid oligosaccharides, and pharmaceutical compositions foralleviating, preventing, or treating obesity, diabetes, and climactericsyndrome, and probiotics for promoting intestinal beneficial bacteria,the pharmaceutical compositions and the probiotics contain thenon-reducing end unsaturated mannuronic acid oligosaccharide as anactive ingredient.

(b) The present invention leads to a production of non-reducing endunsaturated mannuronic acid oligosaccharides, which are active materialsof alginate, and thus provides its excellent antiobesity effect,antidiabetic effect, estrogen activity, and an intestinal microfloracontrolling effect.

(c) These effects are remarkably excellent compared with mannuronic acidoligosaccharides having a non-reducing end saturated mannuronic acidoligosaccharide of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be more apparent from the following detailed descriptiontaken in conjunction with the accompanying drawings, in which:

FIG. 1 shows thin-layer chromatography results of non-reducing endunsaturated mannuronic acid oligosaccharides;

FIG. 2 shows mass analysis results of non-reducing end unsaturatedmannuronic acid oligosaccharides and non-reducing end saturatedmannuronic acid oligosaccharides;

FIG. 3 shows a double bond ratio of non-reducing end unsaturatedmannuronic acid oligosaccharide (USMOS) and non-reducing end saturatedmannuronic acid oligosaccharide (SMOS);

FIG. 4 shows circular dichroism (CD) confirmation results of the sugarcomposition of non-reducing end unsaturated mannuronic acidoligosaccharide (USMOS) and non-reducing end saturated mannuronic acidoligosaccharide (SMOS);

FIGS. 5a and 5b show results of inhibiting lipid accumulation andinhibiting the expression of obesity-related genes aP2, C/EBPα, andPPARγ using real-time PCR, by the treatment with non-reducing endunsaturated mannuronic acid oligosaccharide (USMOS);

FIGS. 6a to 6c show glucose uptake promotion results and expressionlevels of related proteins, p-PAK, p-Akt, and p-AS160, by the treatmentwith non-reducing end unsaturated mannuronic acid oligosaccharide(USMOS);

FIGS. 7a and 7b show intestinal microflora analysis results ofobesity-induced rats by intraperitoneal administration of non-reducingend unsaturated mannuronic acid oligosaccharide (USMOS);

FIG. 8 shows principal coordinate (POC) analysis results of intestinalmicroflora of old mice by the uptake of a non-reducing end unsaturatedmannuronic acid oligosaccharide (USMOS);

FIG. 9 shows phylum-level comparative analysis results of the intestinalmicroflora change of old mice by the uptake of non-reducing endunsaturated mannuronic acid oligosaccharide (USMOS);

FIGS. 10a and 10b show mRNA expression levels of pS2, PR, and CTSD bythe treatment with non-reducing end unsaturated mannuronic acidoligosaccharide (USMOS);

FIG. 11 shows mRNA expression levels of PGC-1α, ERRα, GATA3, and FOXA1by the treatment with a non-reducing end unsaturated mannuronic acidoligosaccharide (USMOS);

FIG. 12 shows a mechanism of non-reducing end unsaturated mannuronicacid oligosaccharide (USMOS); and

FIG. 13 shows various biological activity effects of non-reducing endunsaturated mannuronic acid oligosaccharide (USMOS).

DETAILED DESCRIPTION

Hereinafter, embodiments of the present invention will be describedbelow in detail with reference to the accompanying drawings, so thatthose of ordinary skill in the art may easily work the embodiments.However, the present invention may be realized in various differentforms, and therefore is not limited to embodiments to be describedherein.

EXAMPLE 1 Preparation of Alginic Acid Oligosaccharide

For the preparation of poly-mannuronate (poly M), 1 g of sodium alginate(Wako, Osaka Japan) and 100 ml of 0.3 M HCl were placed together, andheated at 100° C. for 2 hours. The heated sodium alginate-HCl solutionwas centrifuged at 500 g for 5 min, and the separated precipitate wasdissolved in distilled water. After NaCl was added such that theprecipitate dissolved in the distilled water has 0.1 M, the solution wasadjusted to pH of 2.8-3.0, and then centrifuged at 500 g for 5 min toseparate supernatant and precipitate. The separated precipitate andsupernatant were subjected to alcohol precipitation and drying, toprepare poly M from the supernatant and poly G from the precipitate,which were used as substrates for preparing mannuronic acidoligosaccharides using alginate lyase (Haug, A et al. A study of theconstitution of alginic acid by partial acid hydrolysis. Acta ChemicaScandinavica, 1966, 20(1): 183-190., and Joo, D. S. et al. Preparationof oligosaccharides from alginic acid by enzymatic hydrolysis. KoreanSociety of Food Science and Technology, 1996, 28(1): 146-151).

For the preparation of non-reducing end unsaturated mannuronic acidoligosaccharides, a transgenic strain was used, wherein the transgenicstrain was produced by recombining a gene corresponding to ORF11, whichwas selected from metagenomic library of intestine DNA of abaloneinhabiting the sea near Yeosu, Korea in February 2009, and has anexcellent mannuronic acid lysing ability, in pMAL-c2x expression vector,and then cloning it in the BL21(DE3) strain (Korean Patent No10-1277706).

500 ml of the transgenic strain was inoculated in Luria-Bertani (LB)supplemented with 40 L of ampicillin (100 μg/ml), and cultured at 37° C.When the absorbance at 600 nm reached 0.4-0.5,isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to afinal concentration of 0.3 mM, followed by culturing for 12 hours. Onthe completion of the culturing, the culture was centrifuged at 9,000 gfor 15 min to precipitate cells. After the precipitated cells weresuspended in 10 mM phosphate buffer (pH 7.0), sonication was performedfor cell membrane disruption, and centrifugation at 9,000 g for 15 minwas performed for coenzyme isolation. After the centrifugation, theseparated supernatant was used as a coenzyme. 0.3% poly-mannuronate(poly M) was dissolved in 1 L of 10 mM phosphate buffer (pH 7.0), andAgNO₃ was added to a final concentration of 1 mM. A substrate lysisreaction was conducted at 45° C. for 48 h using a 2.5 L-fermentor (KBTKB-250, Japan). After the reaction, the resultant material was filteredthrough an ultrafiltration membrane system (Vivaflow 50, Sartorius, Ag,Germany) to obtain a mixture of oligosaccharides with a molecular weightof 3,000 Da or less, followed by lyophilization, to prepare non-reducingend unsaturated mannuronic acid oligosaccharides.

Thin-layer chromatography was performed to investigate the production ofoligosaccharides, and the method thereof was as follows. Thenon-reducing end unsaturated mannuronic acid oligosaccharides weredissolved in water to a concentration of 0.1 mg/μl, and then 3 μl of thesolution was spotted on silica gel plate (Merck KGaA, Germany). Thenon-reducing end unsaturated mannuronic acid oligosaccharides weresorted by the size thereof using a development solvent (1-butanol:formicacid:water=4:6:1), and then the presence of oligosaccharides wasconfirmed using a color developing reagent (anise aldehyde 0.5 ml,acetic acid 10 ml, MeOH 85 ml, H₂SO₄ 5 ml) added with sulfuric acid.

EXAMPLE 2 Composition and Structural Characterization of Non-ReducingEnd Unsaturated Mannuronic Acid Oligosaccharides

In the present example, poly mannuronate was lysed with AlyDW11 alginatelyase to secure a mixture of the non-reducing end unsaturated mannuronicacid oligosaccharides, from which fractions with a molecular weight of3000 Da or less were then secured using an ultrafiltration membranesystem (VivaFlow 50, Sartorius). In order to analyze component sugars ofthe non-reducing end unsaturated mannuronic acid oligosaccharides, theprepared sample was purified using an ion exchange resin column (HitrapDEAE Sepharose FF, GE Healthcare), followed by lyophilization. Thepurified non-reducing end unsaturated mannuronic acid oligosaccharideswere dissolved in water, and then the solution was injected into UPLC/MSsystem to analyze component sugars thereof.

For setting ultra performance liquid chromatography (UPLC, Waters),ACQUITY UPLC BEH C18 column (1.7 μm 1.0×100 mm, Waters) was used, andthe linear gradient of solvent A (15 mM amylamine and 25 mMhexafluoroisopropanol (HFIP)) and solvent B (15 mM amylamine and 25 mMHFIP in acetonitrile) was controlled at 0.4 ml/min for 12 min. Theeluate separated from C₁₈-UPLC was analyzed using a mass spectrometer(Quadrupole-Time of Flight, Q-TOF, Waters). Q-TOF analysis was carriedout in the ESI negative mode, and the conditions were: capillary andcone voltages were 3 kV and 40 V, respectively; desolvation flow ratewas 600 L/h; temperature was 300° C.; and source temperature was 120° C.TOF MS data was analyzed at a scan time of 0.5 s in the range m/z100-1300. For accurate analysis, 2 ng/μl leucine enkephalin (554.2619 Dain ESI negative mode) was used as a lock spray for all analysis.

As can be seen in FIG. 2, as a result of mass analysis results of thenon-reducing end unsaturated mannuronic acid oligosaccharides, thenon-reducing end unsaturated mannuronic acid oligosaccharides arecomposed of one to seven sugars, and especially, peaks having massvalues, which are smaller than the previously reported mass values ofmannuronic acid oligosaccharides by 18, were observed, and thus it canbe seen that non-reducing end saturated mannuronic acid oligosaccharidesare formed through the removal of a water molecule, and are moredominant than non-reducing end saturated mannuronic acidoligosaccharides (SMOS).

The results showing the ratios of non-reducing end unsaturatedmannuronic acid oligosaccharide (USMOS) to non-reducing end saturatedmannuronic acid oligosaccharide (SMOS) were present in FIG. 3. As shownin FIG. 3, it was verified that the non-reducing end unsaturatedmannuronic acid oligosaccharides had, on average, two times or moremonosaccharides than the non-reducing end saturated mannuronic acidoligosaccahrides. The molecular weights of the non-reducing endsaturated mannuronic acid oligosaccharides are as follows: 1 sugar (m/z193), 2 sugars (m/z 369), 3 sugars (m/z 545), 4 sugars (m/z 722), 5sugars (m/z 898) 6 sugars (m/z 1074), 7 sugars (m/z 1250).

In order to measure the percentage of mannuronic acids in thenon-reducing end unsaturated mannuronic acid oligosaccharides, acircular dichroism (CD) spectroscopy signal was measured using circulardichroism spectroscopy (CD, J-715 spectropolarimeter, JASCO). CD signalswere measured in the region of 190-250 nm using a cuvette (1 cm) at roomtemperature, and in order to obtain consistent CD signals, thenon-reducing end unsaturated mannuronic acid oligosaccharides were usedat 1 mg/ml. In order to investigate the composition ratio of mannuronicacid:guluronic acid, the ratio of mannuronic acid and guluronic acid wascalculated by measuring a peak (an absorbance value at 200 nm) and atrough (an absorbance value at 215 nm). The calculation is as follows:

peak/trough<1, mannuronic acid/guluronic acid=2.0 (peak/trough)   (1)

peak/trough>1, mannuronic acid/guluronic acid=27 (peak/trough)+40   (2)

As can be seen from FIG. 4, it was confirmed that the non-reducing endunsaturated mannuronic acid oligosaccharides had a mannuronicacid/guluronic acid ratio of 2.12 (peak=6.66, trough=6.42).

EXAMPLE 3 Suppression of Non-Reducing End Unsaturated Mannuronic AcidOligosaccharides on Adipocyte Lipid Accumulation

When 3T3-L1 preadiocytes were cultured in DMEM medium to reachconfluence, the cells were treated with 0.5 mM isobutylmethylxanthine(IBMX), 1 mM dexamethasone, and 1 μg/ml insulin (MDI) for 2 days, andthen the medium was exchanged with DMEM+serum medium supplemented with 1μg/ml insulin at an interval of 48 hours, to induce the differentiationinto adipocytes for 7 days. At the time of the exchange of medium, thecells were treated with the non-reducing end unsaturated mannuronic acidoligosaccharides at 0.2 mg/ml. After 7 days, the adipocyte lipidaccumulation and the degree of suppression of differentiation wereobserved by Oil red O staining and RNA extraction, and the results weredepicted in FIG. 5 a.

As can be seen from FIG. 5a , as a result of observing triglycerides,which were stained with Oil red O, using an optical microscope, thetreatment with non-reducing end unsaturated mannuronic acidoligosaccharides at 0.2 mg/ml resulted in a significantly weaker Oil redO staining intensity in the adipocytes, compared with a control.Further, as a quantitative result of triglycerides, which were stainedwith Oil red O, through pigment extraction, USMOS having a double bondat the non-reducing end suppressed triglyceride accumulation by about40%, compared with the control, and non-reducing end saturatedmannuronic acid oligosaccharides suppressed the lipid accumulation byabout 15%, compared with the control. Hence, it was verified that theformation of a double bond is an important factor in the antiobesityeffect.

3T3-L1 preadiocytes were differentiated into adipocytes by the samemethod. The cells were treated with non-reducing end unsaturatedmannuronic acid oligosaccharides at 0.2 mg/ml, and then RNA extractionwas conducted by the GeneJET RNA purification kit, and thereafter, theresults of the inhibition of the expression of aP2, adiposedifferentiation-related gene CAAT enhancer binding protein α (C/EBPα),and peroxisome proliferator-activated receptor γ (PPARγ), which areadipose differentiation markers, were depicted in FIG. 5 b.

As shown in FIG. 5b , it was verified that the treatment withnon-reducing end unsaturated mannuronic acid oligosaccharides reducedthe expression levels of aP2, C/EBPα, and PPARγ by 25%, 60%, and 40%,respectively, compared with the control. In addition, it was verifiedthat the non-reducing end unsaturated mannuronic acid oligosaccharideshad an excellent antiobesity effect, compared with the non-reducing endsaturated mannuronic acid oligosaccharides.

EXAMPLE 4 Verification on Control of Glucose Uptake by Non-Reducing EndUnsaturated Mannuronic Acid Oligosaccharides

L6 muscle cells were cultured in DMEM medium containing 10% serum, andthe L6 cells were completely differentiated while the medium wasexchanged with 2% serum medium. The culture medium containing thecompletely differentiated L6 muscle cells was exchanged with serum-freeDMEM medium, and the cells were treated with non-reducing endunsaturated mannuronic acid oligosaccharides at 0.2 mg/ml for 1 hour.After that, the medium treated with non-reducing end unsaturatedmannuronic acid oligosaccharides was discarded, followed by washing twotimes with previously warmed Krebs-Ringer Hepes buffer (KRH buffer) at37° C., thereby removing glucose in the medium. After the treatment with0.04 mM [³H]-2-deoxyglucose for 15 min, the KRH buffer, which contained[³H]-2-deoxyglucose, was promptly discarded, and then ice-cooled PBS wasadded to stop the reaction. The cells were disrupted using a cell lysisbuffer, and then the radioactivity measurement was conducted using ascintillation counter to investigate a glucose transport abilityincreasing effect by alginic acid oligosaccharide treatment.

As shown in FIG. 6a , it was verified that the treatment withnon-reducing end unsaturated mannuronic acid oligosaccharides promotedthe intracellular glucose uptake to a similar degree, compared withinsulin (0.2 μM) as a positive control. It was verified that, as thetreatment with non-reducing saturated mannuronic acid saccharide at 0.2mg/ml did not lead to glucose uptake, the formation of a double bond atthe non-reducing end was important in the promotion of glucose uptake.In addition, it was verified that, as the co-treatment with non-reducingend unsaturated mannuronic acid oligosaccharides and 1 μM compound C (C.C), which is an inhibitor of AMP-activated protein kinase (AMPK) as animportant protein in glucose uptake, suppressed glucose uptake, thenon-reducing end unsaturated mannuronic acid oligosaccharides promotedthe glucose uptake via AMPK pathway (FIG. 6b ).

In order to investigate an increase in phosphorylation of PAK, Akt, andAS160, which influence the expression of transporters related to thepromotion of glucose uptake, by the treatment with non-reducing endunsaturated mannuronic acid oligosaccharides, the muscle cells weretreated with different concentrations of non-reducing end unsaturatedmannuronic acid oligosaccharides, followed by protein extraction andwestern blotting. As a result, as shown in FIG. 6c , it was verifiedthat the phosphorylation was increased in a dose-dependent manner of thenon-reducing end unsaturated mannuronic acid oligosaccharides.Therefore, it was anticipated that the non-reducing unsaturatedmannuronic acid oligosaccharides would promote the AMPK pathway and thephosphorylation of PAK, Akt, and AS160, for the promotion of glucoseuptake, to influence the expression of glucose transporter 4 (GLUT4).

EXAMPLE 5 Verification on Intestinal Microflora Controlling Efficacy byNon-Reducing End Unsaturated Mannuronic Acid Oligosaccharides

In order to investigate the intestinal microflora improvement efficacyof non-reducing end unsaturated mannuronic acid oligosaccharides, ratswere used as obese animal models and mice were used as aged animalmodels. 3-week aged male SD rats, as obesity-induced rats, werepurchased from Central Lab. Animal Inc, and then acclimatized for 3days. The feeding environment was as follows: temperature was 20±2° C.,relative humidity was 50±10%, light/dark cycle was 12 hours per day, anda high-fat diet was induced for 10 weeks. An experiment was carried outwhile non-reducing end unsaturated mannuronic acid oligosaccharides(0.25 mg/kg) were intraperitoneally administered to experiment groups atan interval of 48 hours. 1-month and 17-month aged male C57BL/6J mice,as old mice, were purchased from Korea Basic Science Institute. Thefeeding environment was as follows: temperature was 20±2° C., relativehumidity was 50±10%, light/dark cycle was 12 hours per day, and anexperiment was carried out for 10 weeks. For experiment groups,non-reducing end unsaturated mannuronic acid oligosaccharides (0.2mg/kg) were supplied to water. After the completion of the experiment,intestine contents of the experimental animals were collected, and 200mg thereof were taken to secure pure DNA using Fast DNA™SPIN Kit forSoil kit on the basis of the method suggested in the kit. Theconcentration and purity of the extracted DNA were measured using aNanodrop, and then the DNA concentration and purity were investigated onthe basis of DNA band results extracted through agarose gelelectrophoresis. For the amplification of bacterial 16S rRNA gene inisolated DNA, amplification PCR was performed using 27F forward primer(GAGTTTGATCMTGGCTCAG) containing V1-V3 hypervariable region and 518Rreverse primer (WTTACCGCGGCTGCTGG) under conditions of initialdenaturation at 94° C. for 5 min and 30 cycles of 30 seconds at 94° C.,45 seconds at 55° C., and 1 minute and 30 seconds at 72° C. PCR productspurified through QIAquick gel extraction kit (Qiagen, Germany) werepyrosequenced using GS Junior Titanium system (Roche, Germany) as a DNAsequencer. Methods and reactions necessary for the pyrosequencing werecarried out by ChunLab (Korea) according to the manufacturer's manuals.

As shown in FIGS. 7a and 7b , in the intestinal microflora of obese ratsreceiving non-reducing end unsaturated mannuronic acid oligosaccharides,Roseburia sp. and Lactobacillus sp., which belong to gram positivebacteria (Firmicutes), were increased by about 4% and 2%, respectively,compared with the control (obesity-induced rats, high-fat diet (HFD)),resulting in the microflora change, and Clostridium sp. and Ruminococcussp., which belong to gram negative bacteria (Bacteroidetes), wereincreased by about 1%, respectively.^(4,5)

As shown in FIG. 8, it was verified through PCO analysis that, in theold mice drinking non-reducing end unsaturated mannuronic acidoligosaccharides, the intestinal microflora thereof was similar to thatof 1-month aged mice, but were different from that of 17-month agedmice. In addition, it was verified that the above old mice formedsimilar intestinal microflora to 1-month aged mice taking non-reducingend unsaturated mannuronic acid oligosaccharides.

As can be seen from FIG. 9, it was verified that, in the 17-month oldmice taking non-reducing end unsaturated mannuronic acidoligosaccharides, gram positive bacteria (Bacteroidetes) were increasedby about 22%, and relatively, gram negative bacteria (Firmicutes) weredecreased by about 22%, compared with a control (17-month aged mice),and these results were similar to the intestinal microflora of the1-month aged mice.

EXAMPLE 6 Verification on Estrogen Sensitizer Function of Non-ReducingUnsaturated Mannuronic Acid Oligosaccharides

17β-estradiol used in the present study was purchased from Sigma (St.Louis, Mo., USA), and Dulbecco's modified Eagle's medium/F12 (DMEM/F12),fetal bovine serum, Opti-MEM medium, and penicillin-streptomycin werepurchased from Gibco (NY, USA). PBS, cell count kit (CCK-8), RNeasysmall kit, bovine insulin, and FuGENE HD were purchased from WeIGENE(Daegu, Korea), Dojindo Molecular Technologies (Tokyo, Japan), QIAGEN(Hiden, Germany), Cell Applications (San Diego, USA), and Promega(Madison, Wis., USA), respectively.

MCF-7 cells were cultured at 37° C. in DMEM/F12 medium supplemented with10% bovine fetal serum, penicillin-streptomycine (100 U/ml), and 1%bovine insulin, and in order to measure estrogen sensitizer activity,the estrogen response element (ERE)-luciferase activity and theexpression levels of pS2, PR, CTSD, PGC-1α, ERR, GATA3, and FOXO1 wereinvestigated.

The cells were treated with non-reducing end unsaturated mannuronic acidoligosaccharides (0.1 mg/ml) for 48 hours, and then, in order toinvestigate ERE-luciferase activity, luciferase analysis was carried outby transfecting the MCF-7 cells with pEGFP-C1-ERα, 3× ERE TATA luc, andpRL-SV40 using FuGENE HD reagent, followed by dissolving. RNA extractionwas carried out by GeneJET RNA purification kit method, and then theexpression levels of pS2, PR, CTSD, PGC-1α, ERR, GATA3, and FOXO1 wereinvestigated through real-time PCR.

As shown in FIGS. 10a and 10b , it was verified that, unlike thenon-reducing end saturated mannuronic acid oligosaccharides, thetreatment with non-reducing end unsaturated mannuronic acidoligosaccharides increased the expression of pS2, which is an estrogensignal underlying gene, by about five times, compared with the control,and the co-treatment with estrogen (E2 and 17β-estradiol) and thenon-reducing end unsaturated mannuronic acid oligosaccharides increasedERE luciferase activity and the expression of pS2 and PR, throughestrogen receptor α (ERα), and reduced the expression of CTSD, and thusthe non-reducing end unsaturated mannuronic acid oligosaccharidesselectively regulated the expression of estrogen receptor α underlyingsignal genes.

As shown in FIG. 11, the treatment with non-reducing end unsaturatedmannuronic acid oligosaccharides increased the expression of peroxisomeproliferator-activated receptor c coactivator-1a (PGC-1α) and itstranscription partner, estrogen related receptor α (ERRα), therebyincreasing mRNA expression of GATA binding protein 3 (GATA3) andforkhead box protein A1 (FOXO1) together with ERα pathway, and thus thenon-reducing end unsaturated mannuronic acid oligosaccharides had anestrogen sensitizer efficacy.

EXAMPLE 7 Mechanism Diagram of Antiobesity, Anti-Diabetic, and EstrogenSensitivity-Increasing Actions of Non-Reducing end UnsaturatedMannuronic Acid Oligosaccharides

The overall diagram of mechanisms of antiobesity, anti-diabetic, andestrogen sensitivity-increasing actions of the non-reducing endunsaturated mannuronic acid oligosaccharides was presented in FIG. 12.

As shown in FIG. 12, as a result of summarizing the mechanisms ofantiobesity, anti-diabetic, and estrogen sensitivity-increasing actionsby the treatment with non-reducing end unsaturated mannuronic acidoligosaccharides, it was verified that the treatment with non-reducingend unsaturated mannuronic acid oligosaccharides increased theexpression of PGC-1α through AMPK activation, and activatedestrogen-related receptor α, β, γ (ERRs), which are transcriptionpartners of PGC-1α, thereby promoting fatty acid β oxidation, and thusthe non-reducing end unsaturated mannuronic acid oligosaccharides had anantiobesity effect. In addition, as intramuscular AMPK has been reportedto promote the fatty acid β oxidation metabolism, by mediating the fattyacid synthesis and degradation, and to increase the expression ofmitochondria-related genes through PGC-1 expression, the non-reducingend unsaturated mannuronic acid oligosaccharides were anticipated toincrease the expression and number of mitochondrial genes through AMPKactivation and by increasing the expression of PCG-1α, and thus thenon-reducing end unsaturated mannuronic acid oligosaccharides had anefficacy of improving insulin resistance.

It was verified that, the non-reducing end unsaturated mannuronic acidoligosaccharides, as an estrogen sensitizer, when used together withestrogen, increased the mRNA expression of GATA3 and FOXO1 through theestrogen receptor α (ERα) pathway, and increased the expression of ERRαand PGC-1α, and thus the non-reducing end unsaturated mannuronic acidoligosaccharide had an estrogen sensitizer function by activating EREthrough the ERα pathway dependent on PGC-1α.

In addition, it was verified that the non-reducing end unsaturatedmannuronic acid oligosaccharides improved the intestinal microflora inthe body, and thus increased antiobesity indicator strains (Roseburiasp. and Lactobacillus sp.) and decreased obesity indicator strains(Clostridium sp. and Ruminococcus sp.), and thus the non-reducing endunsaturated mannuronic acid oligosaccharides had antiobesity,anti-diabetic, intestinal microflora-improving, and estrogensensitivity-increasing efficacies in combination.

REFERENCES

1. Qin J1 et al. A human gut microbial gene catalogue established bymetagenomic sequencing. nature. 2010. 59-65

2. Human Microbiome Project Consortium. Structure, function anddiversity of the healthy human microbiome. nature. 2012. 207-214

3. Turnbaugh P J et al. An obesity-associated gut microbiome withincreased capacity for energy harvest. nature. 2006. 1027-31

4. Nadal I et al. is in clostridia, bacteroides andimmunoglobulin-coating fecal bacteria associated with weight loss inobese adolescents. Int J Obes(Lond). 2009. 758-767

5. Neyrinck A M et al. Prebiotic Effects of Wheat Arabinoxylan Relatedto the Increase in Bifidobacteria, Roseburia and Bacteroides/Prevotellain Diet-Induced Obese Mice. PLoS One. 2011. e20944

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1.-17. (canceled)
 18. A method for alleviating or treating obesity,comprising administering to a subject in need thereof a compositioncomprising, a non-reducing end unsaturated alginate oligosaccharidehaving a molecular weight of 100-3,000 Da, which is obtained by lysingpolymannuronate as a substrate with alginate lyase.
 19. The method ofclaim 18, wherein the non-reducing end unsaturated alginateoligosaccharide reduces the growth of intestinal bacterial strains. 20.The method of claim 19, wherein the intestinal bacterial strains areselected from the group consisting of Roseburia sp. and Lactobacillussp.
 21. The method of claim 18, wherein the composition is apharmaceutical composition for alleviating or treating obesity, or afood composition or functional food composition for alleviating obesity.22. A method for alleviating or treating diabetes, comprisingadministering to a subject in need thereof a composition comprising anon-reducing end unsaturated alginate oligosaccharide having a molecularweight of 100-3,000 Da, which is obtained by lysing polymannuronate as asubstrate with alginate lyase.
 23. The method of claim 22, wherein thenon-reducing end unsaturated alginate oligosaccharide promotes glucoseuptake.
 24. The method of claim 22, wherein the composition is apharmaceutical composition for alleviating or treating diabetes, or afood composition or functional food composition for alleviatingdiabetes.
 25. A method for alleviating or treating climacteric syndrome,comprising administering to a subject in need thereof a compositioncomprising a non-reducing end unsaturated alginate oligosaccharidehaving a molecular weight of 100-3,000 Da, which is obtained by lysingpolymannuronate as a substrate with alginate lyase.
 26. The method ofclaim 25, wherein the composition further comprises 17β-estradiol. 27.The method of claim 25, wherein the non-reducing end unsaturatedalginate oligosaccharide exhibits a synergic effect together with17β-estradiol.
 28. The method of claim 25, wherein the composition is apharmaceutical composition for alleviating or treating climactericsyndrome, or a food composition or functional food composition foralleviating climacteric syndrome.